Expansion Of Rat Mesenchymal Stem Cell In Hollow-fiber Membrane Bioreactor Under Hypoxia Condition

Rat mesenchymal stem cells (MSCs) represent a small portion of the cells in the stromal compartment of bone marrow, capable of differentiating in vitro into mature-like cells from all three germ layers. They have the potential to differentiate into cartilage cells, cardiac muscle cells, adipose cells, nerve cells and osteoblasts. They are the promising "seed cells" in tissue engineering and ideal target cells in gene therapy for their low implanting response, being easy to be transfected and expressed steadily and function of supporting hematopoiesis.However, MSCs are in limited quantity and during the process of proliferation, MSCs may lose the activity of proliferation and differentiation gradually under the normal culture conditions, which can not meet the need of autologous repairation and clinical therapy. So how to reduce the damage during the cell proliferation, and retain the character and the ability of secreting matrix of the stem cells is very important.In order to raise the expansion fold and retain the stemness, a perfusion hollow fiber membrane bioreactor was developed and used to culture and expand MSCs in vitro. SD rat mesenchymal stem cells (rMSCs) were isolated with density gradient centrifugation. The cells were seeded on cellulose acetate hollow-fiber membrane constructs and preincubated for 24 hours. Then the constructs were transferred to the bioreactor and cultured for up to 7 days. Because the partial pressure of the atmospheric oxygen (pO₂) in bone marrow is much lower than that in the air, rMSCs were grown under low-pO2 conditions (5% oxygen) and air (21% oxygen) for comparison. The cell expansion fold, colony formation, multi-differentiation were assayed and fluorescence-activated cell sorter analysis were carried out to examine the role of different pO₂ in dynamic condition on rMSC proliferation.The results showed that in hollow-fiber membrane bioreactor, O₂ concentration kept almost constant; MSCs expansion fold in 5%O₂ is 11, while 8 in control oxygen and proliferation potential and metabolic activity were higher for cultures maintained in low oxygen than those in control oxygen. In 5%O₂ condition rMSCs on hollow-fiber membranes can maintain significantly higher colony-forming unit capabilities than normoxic controls, approximately 112 and 53 respectively.Hypoxic rMSCs also expressed higher levels of CD44⁺ and CD29⁺ markers than normoxic controls. After being induced, the expanded cells still reserved the strong muti-differentiation potential. Therefore, it's safe and effective for MSCs culture and expansion in vitro with the method used here.